Fundamental cellular activities such as the transcription, replication, recombination and repair of genes require the non-covalent interaction of DNA and DNA-binding proteins. The underlying molecular recognition processes governing protein-DNA interactions are complex and not yet fully understood. In particular, critical and substantial gaps exist in our knowledge and understanding of the role played by molecular dynamics in protein/DNA interactions. A general problem in the field of molecular recognition is that structural studies reveal relatively little about the entropic component of the free energy of complex formation. Thus, it is very important to complement available structural information by undertaking studies designed to elucidate details concerning the nature and contributions of conformational dynamics in the protein/DNA interface. We propose to use solution-state NMR spectroscopy to characterize the dynamics of amino acid side chains in model systems of protein/DNA complexes. The model system chosen for study is the so-called K50 class of homeodomain proteins, which is defined as those homeodomains that have a lysine residue at position 50 in the DNA recognition helix. The homeodomain motif has been a very important model system for characterizing protein/DNA interactions, and a wealth of information concerning homeodomain/DNA interactions has been provided by both functional and structural studies. These structural studies have demonstrated a conserved global fold and docking arrangement, but fundamental questions remain concerning the roles of key amino acid residues at the protein-DNA interface and about the extent and significance of side chain motions. The general hypotheses of the proposed research are that modulation (as opposed to immobilization) of protein side chain dynamics plays important roles in (1) establishing a complementary interface between DNA and a cognate DNA-binding protein, and (2) allowing a given DNA-binding protein to recognize consensus and non-consensus DNA sequences. The specific aims of the project are: (1) to determine the side chain dynamics of the homeodomain from the Drosophila Bicoid protein, bound to a DNA duplex containing the consensus binding site TAATCC; (2) to determine the side chain dynamics of the Bicoid homeodomain bound to a non-consensus DNA site; and (3) to determine the structure and dynamics of the Pitx2 homeodomain/DNA complex. Each of these specific aims will require a structure determination of the homeodomain/DNA complex; to date no K50-class homeodomain structure has been reported. Clearly, the homeodomain model cannot be fully representative of all protein/DNA interactions. However, the comprehensive studies of protein dynamics that are proposed herein will help to create a foundation of knowledge upon which future work can build, regarding the nature of protein motions and their significance in protein/DNA recognition.